Apparatus and methods for flow photometry of particles of a dispersion

ABSTRACT

In flow photometry by means of transmitting light from a light source to a flowing dispersion in a measuring zone and from the dispersion to a photosensitive receiver and associated apparatus for measuring and/or counting the particles of the dispersion and in which there is an optical axis extending from the measuring zone to the photosensitive receiver, the improvements in which the dispersion is caused to flow through the measuring zone with a velocity component parallel to the optical axis, and a stream of the dispersion medium optically empty or other optically empty fluid of like refractive index is directed to wash the dispersion from the measuring zone or to form an envelope around the dispersion as the dispersion is introduced into the measuring zone.

Dittrich et a1.

11 3,738,759 June 12, 1973 [54] APPARATUS AND METHODS FOR FLOW 3,413,46411/1968 Kamentsky 356/39 X PHOTQMETRY 0 PARTICLES OF A 2,626,361 1/1953Martine 356/207 DISPERSION 3,541,336 11/1970 Einstein 356/207 X3,462,609 8/1969 Beattie 356/103 X [76] Inventors: Wolfgang Dittrich, AmKrug 42;

' Wolfgang Giihde, Lohafenerweg 39, Primary Examiner-Ronald L. Wibertboth of Muenster, Germany Assistant ExaminerF. L. Evans Attorney-D. C.Roylance, David S. Abrams, [22] Flled' 1970 Robert H. Berdo, Donald A.Kaul, Walter C. Farley PP 2 ,215 and John D. Crane Related U.S.Application Data V [63] Continuation-impart of Ser. No. 884,651, Dec.12, [57] ABSTRACT 1969, abandoned, In flow photometry by means oftransmittmg light from a light source to a flowing dispersion in ameasuring [30] Foreign Application Priority Data zone and from thedispersion to a photosensitive re- Apr. 18 1969 Gennany P 19 19 628.2ceivei and associaied apparatus for measuring and/m counting theparticles of the dispersion and in which [52] CL 356/208 250/218 250/222PC, there is an optical axis extending from the measuring 3 56 /246 zoneto the photosensitive receiver, the improvements 51 1m. 01. 00111 21/26in which the dispersion is caused to flow through the [581 Field ofSearch 356/102, 103 207, measuring Zone with a velocity componentParallel 35 20 3 39 244 24 250 21 2 22 PC the optical axis, and a streamof the dispersion medium i optically empty or other optically emptyfluid of like [56] References Cited refractive index is directed'to washthe dispersion from the measuring zone or to form an envelope around the1 UNITED STATES PATENTS dispersion as the dispersion is introduced intothe mea- 3,493,304 2/1970 Rovner 250/71 R swing zone.

3,609,048 9/1971 Strickler 356/246 3,254,558 6/1966 Gramm 250/222 PC 6Claims, 5 Drawing Figures I0 AMPLIIFIER COUNTER II I2 -h '3 l4 MUL TI-CHANNEL ANALYZER 8 BRAUNIAN TUBE 7 34 A J ii 4 4 X I7 24 .J)'A I VII/LPATENIED JUN 31975 3. 738, ?E9

SHEEI 1 BF 3 I AMPLIIFIER COUNTER ll I2 MUL TI- CHA NNEL ANALYZER 8BRAUNIAN TUBE INVENTORS WOLFGANG DITfR/CH WOLFGANG GOHDE ATTORNEYSPATENFEW'ZW 3138075 SHEEI 2 0f 3 46 COUNTERS COUNTER 34 yaw,

I9 '7 I kg I N VEN TORS WOLFGANG DITTRICH WOLFGANG GHDE BY f 7/I55/ ATTORNE Y3 PATENFED 3. 738,759 sum 3 or a FIG-4 IN VENTORS WOLFGANG DITTRICH WOLFGANG GOHDE APPARATUS AND METHODS FOR FLOW PHOTOMETRY FPARTICLES OF A DISPERSION This is a continuation-in-part of applicationSer. No. 884,651, filed Dec. 12, 1969, now abandoned.

This invention relates to apparatus and methods for the flow photometryof particles of a dispersion, partic ularly to such apparatus providedwith an automatic measuring and counting device and in which allparticles to be measured and counted traverse the depth of focus rangeof a microscope arrangement used. The particles to be measured andcounted may be, for example, blood corpuscles, tumor cells, single-cellalgae, plankton, bacteria, and the like.

It is an object of the invention to provide improvements upon theapparatus described in detail in the aforementioned parent application.

The device more fully described in the aforementioned applicationpermits scanning the particles optically in a rapid flow process; duringthe passage of the particles through a fixed measuring point, specificlight pulses correlated with the particle properties are transformedinto electric pulses, which can then be recorded by means of suitableamplifier and counting devices. In this way, physical, physical-chemicaland chemical properties of a plurality of particles of a certaincollection can be determined simultaneously with the aidof an automaticmeasuring and counting device during flow.

The fixed measuring area of the measuring and counting device describedin said application, at which the particle properties are determined byan optical method, lies within a nozzle aperture which is traversed bythe dispersion approximately in the direction of the optical axis of amicroscope arrangement comprising the apparatus. Nozzles of other designare generally unsuitable for this counting and measuring device, becausespecific optical facts are at hand to which the special design of thenozzle or nozzle aperture must be adapted. Prior art devices in which adispersion stream is conducted transversely to the optical axis do notgive photoelectric current signals quantitatively corresponding to theparticle properties in a unique manner.

According to the present invention, there is provided a device for flowphotometry'of-particles having a measuring area which is not definitelyfixed by therigid walls of a flow chamber and the position of whichresults also fromthe dynamic interaction of the flowing dispersion withone or more streams of optically empty -(i.e., particle-free) dispersionmedium or of another 7 larger particle contributes to the photo currentsignal in a consistent manner without giving preference to a detail ofthe particle traversing the measuring point corresponding to the optimumof the depth of field of the microscope arrangement. in connection withsuitable flow chambers using selectively illumination by transmittedand/or incident light or darkfield illumination and simultaneousdirection of the light stream onto several photomultipliers withelectronic evaluation, a versatile, multiparametric automatic measuringand counting device is provided.

The objects of the invention are attained by, for one thing, theprovision of a specific design for the scanning device or nozzle inconnection with a microscope arrangement; each particle of thedispersion furnishes photoelectric current signals uniquely correlatedto certain particle properties. More particularly, a flow chamber havingcertain characteristics is used in connection with an incident and/ortransmitted light microscope. The essence of the flow chamber used isthat all particles of the dispersion entering the measuring zone of themicroscope arrangement and to be counted and/or measured are forced totraverse the depth of field of the microscope arrangement in aconsistent manner with a velocity component parallel to its opticalaxis. This requirement is fulfilled by the flow chamber described in theaforementioned application in that in the flow chamber of thatapplication the particles are drawn or pushed through a uniformlyilluminated nozzle in the direction of the optical axis of themicroscope, the aperture of which is sharply projected onto themeasuring diaphragm or onto the surface of the photomultiplier.According to the present invention, however, the construction of anozzle serving as measuring point may be wholly or partially dispensedwith when the dispersion is moved in a thin jet, surrounded by anenveloping stream of the optically empty dispersion medium or of anothermedium having the same index of refraction through the measuring zoneprojected completely onto one or simultaneously onto severalphotomultipliers or measuring diaphragms with a velocity componentparallel to the optical axis and, hence, through the entire depth offield of the microscope objective.

The use of an enveloping stream offers in particular the advantage of auniform flow velocity of the dispersion at uniform orientation, broughtabout by hydrodynamic forces, of those particles which greatly differfrom the spherical form. According to the invention, there is alsoprovided with incident light illumination an especially simply designedflow chamber, in which the dispersion issues from a cylindrical channelor a nozzle in the direction of the photo-sensitive'receiver and isconducted away from the measuring area by" a stream of the opticallyempty dispersion medium or another liquid having the same opticalproperties, directed transversely thereto. As compared with prior artarrangements, this has the advantage that no particles of the dispersioncan become caught inside the measuring zone of the flow chamber oradhere to the chamber wall, whereby the measurement result could beadversely influenced. Also here, by suitable adjustment of theillumination, it can be achieved according to the invention that theproportion of dispersed and/or fluorescent light arriving from eachparticle of the dispersion on one or more photomultipliers remainsapproximately constant at a displacement in the direction of the opticalaxis corresponding to the greatest particle diameter, and that thus theamplitude value of the emitted photoelectric current signals furnishesan unambiguous measure'of the particle optical properties.

According to the invention, a flow chamber of the invention is usedtogether with an incident and/or transmitted light microscope and withelectronic devices for the counting and/or recording of the frequencydistributions of the particles of a dispersion. Instead of a completemicroscope, under certain conditions an objective alone may besufficient, the measuring diaphragm or the photo-sensitive receiverbeing arranged in the intermediate image plane". For the determinationof the number of particles per unit volume, the measuring and countingdevice is equipped, according to the invention, with devices whichpermit the simultaneous recording or calculation of the dispersionvolume passing through the flow chamber. In particular, it is intendedto insure a specific rate of flow of the dispersion by means of auniformly working pump or to force or draw a given volume of dispersionthrough the flow chamber.

Preferred embodiments of the invention will now be described inconjunction with the drawings, in which:

FIG. I is a schematic diagram of an entire measuring and counting deviceaccording to the invention;

FIG. 2 is a schematic diagram of an entire measuring and counting deviceaccording to the invention for the simultaneous pick-up of up to threeparameters;

FIG. 3 is a partly broken away isometric view of a flow chamber of theinvention; and

FIGS. 4 and 5 are cross sectional views of two other flow chambersaccording to the invention.

Light from a constant light source 1 passes through a collector lens 2,a field diaphragm 4, an exciter filter 3 and an objective 8' whichserves as condenser lens for light from the source 1 and falls on ameasuring area 7 inside a flow chamber 6 (FIGS. 1 and 2). The flowchamber consists of a base body 22 of corrosionresistant metal or ofglass, for example, with a bore 16 parallel to the optical axis of amicroscope arrangement at least at the measuring point 7. Bore 16 servesas the inflow channel for the dispersion. The microscope arrangementcomprises lenses 8 and 8 defining a microscope objective. In base body22' of the slightly modified embodiment shown in FIG. 3, the inflowaperture and measuring area 7 is located in a shallow, tubshapeddepression 23. There is provided an inflow channel 24 and an outflowchannel 17 for a cross stream. Facing a photo-sensitive receiver(photomultiplier) the depression 23 is covered with a planeparallelplate 19 of light-transmitting material (e.g., glass or quartz).Dispersed light and/or fluorescent light emitted by the particlespassing the measuring point 7 falls, via the lens 8', an optical dividerdisk 34 which serves to illuminate the measuring point 7 with incidentlight from the source 1, the lens 8, an exciter light barrier filter 9and a measuring diaphragm 35, on the photo-sensitive surface 36 of thephotomultiplier 10.

The photoelectric current signals emitted by the receiver 10 areamplified by means of an electronic amplifier 11 and counted by anelectronic counting device 12. A Braunian tube 13 serves to control thephotoelectric current signals. Histograms of the photoelectric currentpulse heights can be obtained with the aid of a multi-channel analyzer14.

In FIG. 2 is illustrated a measuring and counting device forsimultaneous pick-up of up to three mutually independent parameters. Thelight emitted by the particles passes through the lens 8' onto anoptical divider plate 37. A portion of the light then passes through alight filter 9 for the selection of a wave length range, correlated tothe first parameter, of the light emitted by the particle and through ameasuring diaphragm 35 onto the photomultiplier 10. From the dividerplate 37 part of the remaining light passes through a second dividerplate 38 onto a photomultiplier 39 and the other part is transmitted bythe divider plate 38 to a photomultiplier 40. Before photomultipliers 38and 40 are respective light barrier filters 41 and 42 for the selectionof the wave length ranges, correlated to the second and thirdparameters, of the light emitted by the particle, and respectivemeasuring diaphragms 43 and 44. The photoelectric current signalsobtained from each photomultiplier are counted as described for FIG. 1and/or recorded in accordance with the amplitude value, either of whichtype of apparatus is schematically illustrated by the boxes 45, 46 and47. With the aid of a uniformly working pump 48 a constant rate of flowof the dispersion through the flow chamber is achieved; thedetermination of the number of particles per unit volume is thuspossible.

FIG. 4 is another construction of a flow chamber in which the dispersionstream is first guided parallel to the optical axis of the microscope.The dispersion passes through a capillary tube 28 to the beginning of anarrow channel 29; the dispersion stream 49, which is surrounded by anenveloping stream 50, flows through the narrow channel 29, which opensat 7 into a wide channel 25 extending at right angles to the opticalaxis. The object plane 0 of the microscope is at the point of confluence7 or a little thereabove. Through the aperture 51 the fluid toconstitute the enveloping stream is admitted. This is a fluid not havingany particles dispersed therein (optically empty) and having the sameindex of refraction as the dispersion medium. Through the aperture 26,likewise optically empty fluid again with the index of refraction of thedispersion medium is admitted as the cross stream 44 which functions tocarry the dispersion very rapidly from the measuring area 7 off to theside.

The two or, when using an enveloping stream, three liquid componentstraversing the chamber are conducted away or sucked off through theaperture 27. Through the light-transmitting, planar plate 19 themeasuring area 7 is illuminated according to the incident light method,and at the same time the dispersed and/or fluorescent light signalsemitted by the particles pass through the cover plate 19 into themicroscope.

When not needed, the enveloping stream, which standardizes the rate offlow of the particles of the dispersion and also, in the case of agreater deviation of the particles from the spherical form, favorsuniform orientation of the particles with the largest diameter in thedirection of flow, may be dispensed with. For this purpose, the inlet 51is not employed. The narrow channel 29 is then traversed only by thedispersion, which is conducted away from the measuring point 7 above thepoint 7. 1

FIG. 5 illustrates a flow chamber in which the formation of a fixedmeasuring point is dispensed with. This flow chamber consists of aplanar light-transmitting base plate 30, a cavity 52 filled with anoptically empty, liquid medium, and a likewise planar,light-transmitting cover plate 19. The inflow of the dispersion occursthrough a capillary tube 32 tapering toward the interior 52 of the flowchamber. The optically empty liquid for the enveloping stream isadmitted through an annular channel 33 which envelopes the capillarytube 32. The mutually parallel channels for the enveloping stream 33 andfor the dispersion stream 32 are disposed on the side wall 53 of theflow chamber in such a way that the dispersion stream forms with theoptical axis A of the microscope an angle differing from The dispersionevaluation.

stream then completely traverses the measuring range 31, provided inchamber 52 by focussing the microscope onto some plane between 32 and17, and traverses it in an oblique direction having a component whichcoincides with the direction of the optical axis. Opposite the dischargeof the dispersion into the flow chamber is a drain 27, through which thedispersion together with its enveloping stream is drawn off. To attainas high as possible a light density in the measuring plane 3l,the fielddiaphragm is projected by means of a condenser into the measuring planein range 31 by the incident and/or transmitted light method. That theangle between the dispersion stream and the optical axis of themicroscope differs from 90 assures that the dispersion stream traversesthe measuring range, that is or the entire depth of field of themicroscope, projected onto the photomultiplier or the measuringdiaphragm, and traverses it with a velocity component parallel to theoptical axis. it is thereby also achieved thatevery particle of thedispersion traverses the depth of field of the microscope in uniformmanner. The dispersed and- /or fluorescent light emitted by eachparticle upon passage through the measuring point passes through thelight-transmitting cover plate 19 and through the microscopearrangement, in the manner already described, to one or morephotomultipliers, which furnish the photoelectric current signals neededfor electronic What is claimed is: 1. A method of flow photometry ofparticles ofa fluid dispersion comprising:

focusing a beam of light to a focal plane of microscopic depth tohomogeneously illuminate a measuring region within said focal plane;passing a particle carrying first fluid dispersion through said plane ina direction having a component perpendicular to said focal plane;continuously passing a second fluid past the focal plane in a directionto wash the dispersion from said focal plane; directing light emanatingfrom particles only in said focal plane to a photodetector; andanalyzing a characteristic of the dispersion using the informationderived from the photodetector. 2. A method according to claim 1,further including forming an envelope of a third fluid about thedispersion as his passed into said measuring region.

3. A method of flow photometry of particles ofa fluid dispersioncomprising:

focusing a beam of light to a focal plane of microscopic depth tohomogeneously illuminate a measuring region within said focal plane;

passing a particle carrying first fluid dispersion through said plane ina direction having a component perpendicular to said focal plane;

forming an envelope of a second fluid about the dispersion as it ispassed into the measuring re- 4. An apparatus for flow photometry ofmicroscopic particles in a fluid dispersion comprising the combinationof:

a flow chamber having a transparent wall;

a light source;

an optical system including means for focusing a beam of light to afocal plane of microscopic depth within said chamber to homogeneouslyilluminate a measuring region within said focal plane, and

means for detecting light emanating only from particles passing throughsaid focal plane and for producing a signal usable to analyze acharacteristic of said dispersion;

means for conducting a first fluid dispersion current through said focalplane with a velocity component normal to said focal plane; and

means for continuously conducting a second fluid past said focal planeto remove particles which have passed through said focal plane.

5. Apparatus according to claim 4 wherein said means for conducting afluid dispersion includes first and second tubes having concentric endportions, said first tube being within said second tube and carrying thedispersion current in the direction of the focal plane, and

said second tube providing an enveloping current of fluid around saiddispersion as said particles move through said focal plane.

6. An apparatus for flow photometry of microscopic particles in a fluiddispersion comprising the combination of:

a flow chamber having at least one transparent wall and filled withfluid;

a light source;

an optical system including means for focusing a beam of light to afocal plane of microscopic depth within said chamber to homogeneouslyilluminate a measuring region within said focal plane, and

means for detecting light emanating only from particles passing throughsaid focal plane and for producing a signal usable to analyze acharacteristic of said dispersion; and

means for conducting a fluid dispersion current through said focal planewith a velocity component normal to said focal plane,

wherein said means for conducting a fluid dispersion includes a firsttube having an end spaced from said focal plane for conducting anenveloping fluid,

a second tube located within said first tube and having an end spacedfrom said focal plane for directing a dispersion current towards saidfocal plane said first tube providing an enveloping current of fluidaround said dispersion current as said particles approach said focalplane, and

a third tube to permit said fluids to leave said chamber.

1. A method of flow photometry of particles of a fluid dispersioncomprising: focusing a beam of light to a focal plane of microscopicdepth to homogeneously illuminate a measuring region within said focalplane; passing a particle carrying first fluid dispersion through saidplane in a direction having a component perpendicular to said focalplane; continuously passing a second fluid past the focal plane in adirection to wash the dispersion from said focal plane; directing lightemanating from particles only in said focal plane to a photodetector;and analyzing a characteristic of the dispersion using the informationderived from the photodetector.
 2. A method according to claim 1,further including forming an envelope of a third fluid about thedispersion as it is passed into said measuring region.
 3. A method offlow photometry of particles of a fluid dispersion comprising: focusinga beam of light to a focal plane of microscopic depth to homogeneouslyilluminate a measuring region within said focal plane; passing aparticle carrying first fluid dispersion through said plane in adirection having a component perpendicular to said focal plane; formingan envelope of a second fluid about the dispersion as it is passed intothe measuring region; directing light emanating from particles only insaid focal plane to a photodetector; an analyzing a characteristic ofthe dispersion using the information derived from the photodetector. 4.An apparatus for flow photometry of microscopic particles in a fluiddispersion comprising the combination of: a flow chamber having atransparent wall; a light source; an optical system including means forfocusing a beam of light to a focal plane of microscopic depth withinsaid chamber to homogeneously illuminate a measuring region within saidfocal plane, and means for detecting light emanating only from particlespassing through said focal plane and for producing a signal usable toanalyze a characteristic of said dispersion; means for conducting afirst fluid dispersion current through said focal plane with a velocitycomponent normal to said focal plane; and means for continuouslyconducting a second fluid past said focal plane to remove particleswhich have passed through said focal plane.
 5. Apparatus according toclaim 4 wherein said means for conducting a fluid dispersion includesfirst and second tubes having concentric end portions, said first tubebeing within said second tube and carrying the dispersion current in thedirection of the focal plane, and said second tube providing anenveloping current of fluid around said dispersion as said particlesmove through said focal plane.
 6. An apparatus for flow photometry ofmicroscopic particles in a fluid dispersion comprising the combinationof: a flow chamber having at least one transparent wall and filled withfluid; a light source; an optical system including means for focusing abeam of light to a focal plane of microscopic depth within said chamberto homogeneously illuminate a measuring region within said focal plane,and means for detecting light emanating only from particles passingthrough said focal plane and for producing a signal usable to analyze acharacteristic of said dispersion; and means for conducting a fluiddispersion current through said focal plane with a velocity componentnormal to said focal plane, wherein said means for conducting a fluiddispersion includes a first tube having an end spaced from said focalplane for conducting an enveloping fluid, a second tube located withinsaid first tube and having an end spaced from said focal plane fordirecting a dispersion current towards said focal plane said first tubeproviding an enveloping current of fluid around said dispersion currentas said particles approach said focal plane, and a third tube to permitsaid fluids to leave said chamber.